This invention relates in general to separation processes and apparatus. More specifically, it relates to a chromatographic gel contactor and a related method for recovering a product from a process fluid produced in a bio-reactor.
In certain biotech applications a product such as a protein or enzyme is produced by cells in a bio-reactor. The cells are immersed in a process fluid and the product is released into the fluid. While the use of naturally occurring or biologically engineered cells to produce a specific product is a well known commercial technique, difficulties in separating the product from the process fluid and undesired material carried by the fluid "downstream" of the bio-reactor significantly raise the cost of manufacturing the product. This cost of downstream processing limits the utilization of this approach in the manufacture of biological products, as compared to artificial, chemical production of the products.
Conventional downstream processing uses packed column chromatographic gel technology almost exclusively. Prefiltering of the fluid media is both extensive and necessary in order to remove suspended solids such as mammalian cells or precipitated proteins which ca clog the packed gel and interfere with the recovery of the product. In short, packed column technology requires that the medium carry the product be highly filtered before it reaches the packed column. In packed column technology it is also necessary to control the feed chemistry to obtain the optimal efficiency of binding between the product and the gel beads of the ion-exchange or affinity resin used in the contactor. Solutions to optimize the conditions for bonding the product to the gel are mixed with the feed in special tanks external to the packed column and the mixture then fed to the packed column, typically in a plug flow.
Another significant consideration is that the entire downstream processing should preferably be maintained in an aseptic condition. This filtration is not a sterile step because the filter membrane cannot be steam sterilized. Therefore, to prevent biological organisms that may grow on the filter from traveling back to and contaminating the bio-reactor, it is necessary to use a large tank that can hold the entire batch of the liquid being downstream processed. This harvest tank must be sterilized, and the tank itself is costly. A particular problem is that while cells are held in this tank, and when they are pumped through the filtration system, they tend to die or burst and release DNA into the process fluid. This is of particular concern if the cells producing the product are the result of recombinant DNA technology. Recombinant DNA must be thoroughly removed from the product to meet the standards of the U.S. Food and Drug Administration (FDA). Burst cells also release protein contaminants, including proteases which may inactivate the product.
Another consideration in downstream processing is that gels can have a significant cost. An inexpensive gel costs $200/liter and more expensive, but not uncommon, gels cost $10,000/liter, and up to $100,000/liter. With packed column technology, there is no mechanical attrition of the gel and the gel can be used and reused for long periods of time. Therefore, while mechanical stirring arrangements are known, such as a stirred system manufactured by Pharmacia, heretofore there has been concern over loss of gel due to the mechanical impact and stress on the gel caused by the stirring. In addition, the Pharmacia stir contactor 1) is not continuous feed--it operates on a batch mode with the gel settling after stirring for a packed column like processing once settled--and 2) there is no control against the gel and other particulates clogging the filter if the unit were operated with continuous feed.
Current packed column chromatographic techniques typically involve the introduction of "plugs" of fluids through the gel, such as elution buffer solutions to strip the product from the gel, wash and rinse solutions, and fluids to adjust the pH. In plug flow, ideally the solution is introduced gently and advances through the column uniformly, albeit at a comparatively slow rate, e.g. 80 to 300 cm/hr. The speed of the solution through the column depends on the binding kinetics (the strength and speed of the reaction between the product and the gel) and the hydraulics of the packed column. In a conventional packed column these two issues are interrelated and they together determine the production speed of the column. To increase the production capabilities, commercial units have assumed very large diameters resulting in a "pancake" configuration. There are difficulties in producing plug flow characteristics across this large diameter unit and such devices are inherently not compact. And as noted above, the introduction of these conditioning fluids has typically required a special tank and mixer with associated conduits, valving, and other controls. Such arrangements increase the cost of the apparatus and the cost of the downstream processing generally.
In biotech applications, filtration systems should minimize damage to the product, gel, and cells, and should operate at a commercially viable rate. A significant problem is the piling up of particles in front of the filter, a phenomenon known as polarization. A common solution to this problem is to use a cross flow that sweeps the particles away. This technique is common in spiral wound or hollow fiber filters. However, pumps normally used in such systems to maintain high cross-flow velocities have the drawback of breaking open cells, thereby releasing DNA. They also may require rotary seals which provide a path of entry for bacteria into the processing system. Another approach involves a cylindrical-shaped filter rotating inside a fixed mating cylinder where a set of ring-shaped vortices, known as Taylor vortices, will be induced in the gap between the filter and the stationary cylinder to provide the mechanism of generating the stirring motion of the fluid to clear up the polarization. WFI seals are known and can provide a high level of protection, but they add to the cost of the system and for the requisite degree of protection against bacterial invasion, a holding tank must nevertheless be used. Direct drive rotary filters, utilizing Taylor vortices for anti polarization effect, are marketed by the Membrex company in the United States and by Sulzer Bros. Ltd. in Switzerland. The Membrex system involves the use of an inward flowing rotary filter cartridge driven to rotate by a magnetic drive coupling to avoid one rotary seal. However, the flow passage of the filtrant from the inside volume of the container still must be contained by a rotary seal to have a total sealed system.
A rotary filter can sustain much higher flow rate per unit area then the cross flow types. However, in either the propeller stirrer types, or the rotating cylinder types of filters, a rotary motion is involved with the need of some form of rotary seal which is objectional for biotech applications.
Another drawback of rotary agitated filtration equipment is the progressive reduction of the surface/volume ratio as the size of the equipment is increased.
It is therefore a principal object of this invention to provide an apparatus and a process for selectively recovering a biological product from a process fluid which may contain cells and other particulate matter with a high degree of efficiency and a comparatively short residence time.
Another principal object of the invention is to provide an apparatus that filters particulate matter from the process fluid, retains gel beads, and provides a thorough mixing of the process fluid and the gel with a minimum of attrition to the gel beads.
Another principal object is to provide a system with the foregoing advantage that processes the fluid aseptically and has no rotary seals or other like points of entry of biological contaminants.
Another principal object is to operate within the sterile envelope which surrounds the bio reactor to avoid using a containment vessel to protect against back contamination to the bio-reactor.
A further object is to decouple the binding kinetics from the packed column hydraulics to allow more economical contactor shapes.
A still further object is to provide an apparatus and method with the foregoing advantages with significant capital and operating cost advantages over conventional packed column technology, both in terms of the apparatus required its sterilization and operation, and the labor required in pretreatment of the process fluid.
A further object is to avoid contaminating the feed fluid with DNA or proteins by avoiding equipment that tends to lyse the cells.
Another object is to provide an apparatus which may be readily scaled in size without significant cost or performance disadvantages.
Another object is to provide an apparatus and method which a facilitates rapid change in the chemistry of the product recovery process without special tanks and mixing apparatus.
A further object is to provide an apparatus and a method with the foregoing advantages which can utilize a standard laboratory shaker table as a principal drive to react small volumes of fluids.